Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase from Streptomyces hygroscopicus

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  • SUGIMOTO Naohisa
    Faculty of Life and Environmental Sciences, University of Tsukuba
  • TAKAKURA Yasuaki
    Faculty of Life and Environmental Sciences, University of Tsukuba
  • SHIRAKI Kentaro
    Division of Applied Physics, Faculty of Pure and Applied Sciences, University of Tsukuba
  • HONDA Shinya
    National Institute of Advanced Industrial Science and Technology (AIST)
  • TAKAYA Naoki
    Faculty of Life and Environmental Sciences, University of Tsukuba
  • HOSHINO Takayuki
    Faculty of Life and Environmental Sciences, University of Tsukuba
  • NAKAMURA Akira
    Faculty of Life and Environmental Sciences, University of Tsukuba

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  • Directed Evolution for Thermostabilization of a Hygromycin B Phosphotransferase from <i>Streptomyces hygroscopicus</i>

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説明

To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.

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